Innovative nanopore sequencing technology

ABSTRACT

Methods and apparatus for long read, label-free, optical nanopore long chain molecule sequencing. In general, the present disclosure describes a novel sequencing technology based on the integration of nanochannels to deliver single long-chain molecules with widely spaced (&gt;wavelength), ˜1-nm aperture “tortuous” nanopores that slow translocation sufficiently to provide massively parallel, single base resolution using optical techniques. A novel, directed self-assembly nanofabrication scheme using simple colloidal nanoparticles is used to form the nanopore arrays atop nanochannels that unfold the long chain molecules. At the surface of the nanoparticle array, strongly localized electromagnetic fields in engineered plasmonic/polaritonic structures allow for single base resolution using optical techniques.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Nonprovisional applicationSer. No. 16/215,139 filed on Dec. 9, 2018, which is a continuation ofU.S. Nonprovisional application Ser. No. 15/039,825 filed on May 26,2016, now U.S. Pat. No. 10,184,930 issued on Jan. 22, 2019, which is aU.S. National Stage Application of PCT/US2014/067764 filed on Nov. 26,2014, which claims the benefit of U.S. Provisional Application Ser. No.61/909,116 filed on Nov. 26, 2013, all of which are incorporated byreference herein in their entireties.

BACKGROUND

The human genome is diploid, and a genome sequence is not completeunless all polymorphisms or variants are phased and assigned to specificchromosomes. Additionally, the entire chromosome landscape must bedecoded, including complex structural variants in the genome (i.e.,an—euploidy, translocations, inversions, duplications, loss ofheterozygosity, etc). For example, balanced translocations occur inapproximately 1 in 500 individuals, trisomy 21 occurs in as many as 1 in650 live births, and extensive genome instability occurs in manycancers. Accordingly, complete genome sequencing must be able toidentify all complex genome variants.

There are a number of ultra-high-throughput sequencing technologiesavailable (e.g., Illumina/Solex, SOLiD, Roche/454, PacBio, Ion Torrent,etc.) and under development [e.g., ZS Genetics, IBM GE (U.S. Pat. No.7,264,934), Oxford Nanopore, Noblegen, Bionanomatrix, and GnuBIO. Whilethe cost of sequencing has decreased dramatically, the technology isstill unable to completely sequence a human genome. There remainnumerous regions of the human genome that are still not sequenced in theGRCh37 version of the genome, which consists of 249 scaffolds(http://www.ncbi.nlm.nih.gov/projects/genome/assembly/grc/data.shtml).Additionally, all current commercial technologies require a referencegenome for a high quality assembly. While de novo genome assemblies arepossible with short read technologies, the quality is low relative toresequencing projects. These problems limit the ability of nextgeneration sequencing platforms to identify certain variants, such aslarge structural changes and repeated regions.

High throughput, long-read sequencing technologies are essential forresolving the complexities of the human genome. The human genome isdiploid, meaning there are two copies each of 22 autosomes and twocopies of the sex chromosomes (XX or XY). Long reads are essential tophase the genetic variants that are unique to each of the homologouschromosomes. Additionally, repetitive regions in the genome makesequencing impossible with short reads.

Recent advances in next generation sequencing technologies, along withthe development of robust analytical methods, have given researchers theability to determine the role of sequence variations in a variety ofhuman diseases. However, the vast majority of these approaches produceresults that are limited to finding polymorphisms while neglecting theimportance of haplotypes. Today the most commonly studied variations aresingle-nucleotide polymorphisms (SNPs) and small insertions anddeletions (InDels). This is because current generation sequencingmethods, while proficient in identifying heterozygous loci, are unableto assign polymorphisms to one of the two homologous chromosomes, thuscomplicating the search for gene/disease associations. The HapMap andother projects are developing a haplotype map, but new approaches arerequired to address the cis and trans relationships in variants thatoccur in rare genotypes (e.g., novel somatic mutations) or in alteredgenomes (e.g., cancer).

The lack of haplotype information obtained from current sequencingapproaches limits scientists' ability to draw important biological andmedical conclusions, namely, because lists of polymorphisms areclassified as homozygous or heterozygous, they neglect the importance ofthe context of each polymorphism. As a consequence, researchers oftenfocus only on the variants that occur in protein coding regions (theexome), since only their importance can be predicted. Without thecontext of knowing whether variants in intergenic regions are linked incis and/or through long-range chromatin interactions to affected genes,it is not possible to predict whether such variants are detrimental. Theprincipal advantage of haplotype resolved sequencing over standard wholegenome sequencing (WGS) is that all polymorphisms are assigned to aspecific chromosome (e.g., maternal vs. paternal), and links areestablished between mutations (or variants) in distant regulatoryelements and cis-linked genes on the same chromosome.

The limitations associated with direct haplotype sequencing primarilyrevolve around the relatively short read-length and ‘phaseinsensitivity’ of the current platforms. There have been a fewapproaches to generate haplotype resolved sequence, but these are notconsistent with the $1,000 genome goal, due to the complexity andadditional cost associated with the processes upstream of sequencing.

Nanopore DNA sequencing technologies are attractive since they offerdirect access to the DNA sequence information without amplification orcomplex post processing of the sequence information, and hold thepromise of long reads at high speed. There is a long history of researchand development in various nanopore technologies. However, the promisehas yet to be fully realized, and—in fact—no reads other than ofspecially constructed test DNA samples have been reported. (check withJeremy on this statement) Additionally, single base resolution has notbeen reported with nanopore technologies. The issues identified inprevious research include:

1. Transduction speed of ˜1 base/μs (requiring high bandwidth electricaldetection with concomitant noise and statistical fluctuation issues).

2. Longitudinal resolution greater than single base (typically ˜4 basesfor biological pores)³⁵.

3. Massively parallel application is difficult with electrical readoutmechanisms.

Both biological and solid-state nanopore technologies have beeninvestigated. For biological systems α-haemolysin andgenetically-engineered MspA are the most common nanopores, and varioustechniques to slow the DNA translocation have been demonstrated,involving the use of enzymes or modification of the ssDNA strand to beinterrogated with regions of dsDNA or other disturbances to slow thetranslocation. However, the difficulty associated with the large numberof bases within the nanopore remains.

For solid-state pores the most common materials are silicon nitride andsapphire using ion- or electron-beam technologies to form the nanoscalepores. Graphene is another material that is attracting much attention.Atomic layer deposition can be used post-lithography to refine the poredimensions. Hybrid technologies, adding biological structures tosolid-state pores have also been investigated. Notwithstanding all ofthis activity, the promise of nanopore technology has yet to beachieved.

One final issue with all of these approaches is the need to scale tomassively parallel applications with cost-effective fabrication. Presentfabrication approaches are dominated by direct-write technologies(electron-beam and ion-beam lithographies), which are not scalable tomassively parallel architectures, nor compatible with widespreadadoption of the technology at low cost. Electrical measurements are noteasily scaled to parallel measurements in an ionic fluid environment,optical measurements provide the most promising route to parallelism—theissue is providing the necessary single base resolution.

SUMMARY

The present disclosure provides methods and apparatus for long read,label-free, optical nanopore long chain molecule sequencing. In general,the present disclosure describes a novel sequencing technology based onthe integration of nanochannels to deliver single long-chain moleculeswith widely spaced (>wavelength), ˜1-nm aperture “tortuous” nanoporesthat slow translocation sufficiently to provide massively parallel,single base resolution using optical techniques. A novel, directedself-assembly nanofabrication scheme using simple colloidalnanoparticles is used to form the nanopore arrays atop nanochannels thatunfold the long chain molecules. At the surface of the nanoparticlearray, strongly localized electromagnetic fields in engineeredplasmonic/polaritonic structures allow for single base resolution usingoptical techniques. Surface Enhanced Coherent Anti-Stokes RamanSpectroscopy (SECARS) is one such technique that has the advantage ofnot requiring labeling of the bases. Fluorescence techniques withlabeled bases provides an alternative possibility.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustration of an exemplary method of nanochannelfabrication.

FIG. 2 is an SEM image showing a photoresist pattern for formingnanochannels having a 1 μm pitch.

FIG. 3 is an SEM image showing 1D enclosed channels formed using thetechniques described herein.

FIG. 4 is an SEM image of 500 nm wide channel walls formed by 50-nmdiameter silica nanoparticles.

FIG. 5 is an SEM image of 100 nm wide channels formed using thetechniques described herein.

FIG. 6 is an SEM image showing a multi-layered nanochannel formed usingthe techniques described herein.

FIG. 7 is a schematic illustration of a structure as described hereinincluding a porous barrier interrupting the nanochannels.

FIG. 8 is a top-wise schematic illustration of sample flow through thenanochannels of FIG. 7 .

FIG. 9 is a side view of sample flow through a nanochannel of FIG. 7 .

FIG. 10 is a high resolution SEM image of a nanochannel roof fabricatedusing the methods provided herein.

FIG. 11 is a high resolution SEM image of the roof of FIG. 10 roof afterdeposition of Si₃N₄ CVD layer (partially etched on the right side toform the reservoir). The white circles mark pores that are justappearing as the etch progresses.

FIG. 12 is a schematic illustration of a nanochannel structure asdescribed herein demonstrating liquid penetration through the roof overa barrier formed in the nanochannel.

FIG. 13 is an image of a sample with nanochannels and one 3 μm barrier.

FIG. 14 is an image showing drops of fluid along three 3-μm widebarriers with an electric field applied.

FIG. 15 is a schematic illustration of a nanopore structure of thepresent disclosure with at least one manufactured nanopore assembled onthe roof of the tortuous nanopore structure.

FIG. 16 is a schematic illustration of an embodiment wherein a tortuousnanopore structure is applied to an existing nanopore structure.

FIG. 17A is an image showing the fluorescence from a drop of buffer/λDNAplaced on top of the roof of an HfO₂ ALD nanochannel approximately 15seconds after the drop was placed on the roof.

FIG. 17B is an image of the drop of FIG. 17A 5 minutes after the dropwas placed on the roof.

FIG. 17C is an image of the drop of FIG. 17A 10 minutes after the dropwas placed on the roof.

FIG. 18 is a graph showing Spectra offset for visibility. The fourdotted lines mark unique spectral identifiers. (Graph is modified from aversion shown in Ref. 57)

FIG. 19 is a schematic illustration of a detection scheme according tothe present disclosure wherein the pump and Stokes excitation beams aregenerated with a Ti:sapphire laser and a nonlinear process.

FIG. 20A depicts an exemplary method of DNA manipulation according to anembodiment of the disclosure.

FIG. 20B is a close up of the structure shown in FIG. 20A to better showthe location and presence of the Metal Insulator Metal (MIM) structure.

FIG. 21 is a schematic illustration of a typical experiment where aDNA-containing solution is directly applied to the entrance of ananochannel array.

FIG. 22 is a schematic illustration showing DNA penetration in thenanochannel of FIG. 21 .

FIG. 23 is an image of DNA entering the nanochannel when voltage isapplied.

FIG. 24 is an image of DNA moving out of the nanochannel when thevoltage is reversed.

FIG. 25 is an image showing that an electric field applied in thedirection of DNA movement stretched the molecules towards the positiveelectrode over many 10's of μms.

FIG. 26 is an image showing that an electric field applied in theopposite direction (from FIG. 25 ) compressed the DNA to 2 μm.

FIG. 27 shows data demonstrating the movement and stretching of DNA thatcan be achieved using the herein described nanochannels by applyingdifferent potentials across the device.

FIG. 28 is a schematic illustration of two-tiered nanochannelsengineered with the different tiers positioned in orthogonal directions.

FIG. 29 is an image showing λDNA diffusion between levels of atwo-tiered nanochannel structure.

FIG. 30 is a side view of a nanochannel according to an embodiment ofthe present disclosure.

FIG. 31 is an image showing DNA accumulation at a barrier within ananochannel.

FIG. 32 is an image showing movement of DNA through a barrier.

FIG. 33 is a schematic illustration of an electrical design that couldbe used with the structures described herein.

FIG. 34 is an image of a possible lab-on-a-chip design incorporating thenanochannels and other structures described herein.

DETAILED DESCRIPTION

According to a first embodiment, the present disclosure provides ananochannel including a system of tortuous nanopores having a partiallysealed porous roof. According to another embodiment, the nanochannelfurther comprises an integrated metal-insulator-metal (MIM) plasmonic orplaritonic structure that enhances optical detection of detectableelements within a sample and provides the necessary spatiallocalization. According to yet another embodiment the present disclosureprovides methods and apparatus for long read, label-free, opticalnanopore long chain molecule sequencing. Suitable long chain molecules(sometimes referred to herein as “molecules” or “molecules of interest”)include DNA, RNA, proteins, etc. Of course it will be understood thatwhile various embodiments and examples may make reference to a specifictype of long chain molecule, such as DNA, unless otherwise specificallystated, the present disclosure contemplates that such embodiments andexamples are similarly applicable to other types of long-chain moleculesincluding, but not necessarily limited to RNA and proteins.

According to some embodiments, the sequencing technology describedherein may be capable of sequencing a full human genome in under one dayfor a cost of ˜$100. According to various embodiments, the technologydescribed herein make use of one or more of: an integrated system ofnanochannels; tortuous (extended and convoluted) nanopores at aseparation greater than an optical wavelength; a metal-insulator-metal(MIM) plasmonic or polaritonic structure, or other optical detectionenhancement structure as described herein; and an optical readoutmechanism such as surface-enhanced coherent anti-Stokes Raman scatteringor labeled fluorescence techniques.

According to still further embodiments, the present disclosure providesmethods for making each of the above. According to some embodiments,only a single, straightforward lithography step, at an easily accessedpitch of ˜1 μm is required. According to various embodiments, nanoscalefeatures are produced by directed self-assembly processes making this aninexpensive and field-replaceable technology.

According to various embodiments, the present disclosure utilizesnanochannels formed from nanoparticles. According to an embodiment,self-assembled nanochannels can be formed by directed spin-coating ofnanoparticles (˜50 nm diameter or less) onto photoresist walls, formedby a sequence of lithography steps that include some appropriate variantof exposure, development and etching as is well known in the art, suchthat the nanoparticles are “stacked up” to form the nanochannel wallsand roofs. Suitable materials for forming the nanoparticles includematerials for which a method to remove the photoresist exists.Furthermore, it will be understood that in those embodiments wherein thenanochannel is to be used with nucleic acids, the material should behydrophillic to enable filling of the nanopores with a solution andnegatively charged to enable transloction of the nucleic acids throughthe nanopores. According to a specific embodiment, silica nanoparticleshave been found to meet all of the above-identified requirements. Thespin-coating step is followed by a “lost-wax” calcination step thatburns out the photoresist, sinters the nanoparticles to providemechanical strength, and provides a hydrophilic surface for fluidintroduction. Alternate processes such as solvent removal can be used toremove the photoresist and the ARC layers. Additional details for theformation of such nanochannels can be found in U.S. Pat. No. 7,825,037,which is hereby incorporated by reference.

Turning now to FIG. 1 , which is a schematic illustration of anexemplary method of nanochannel fabrication, it can be seen that in thedepicted embodiment, nanochannel fabrication includes multiple steps.First, a substrate (for example, quartz or fused silica) is spin-coatedwith a bottom antireflection coating and then a photoresist layer. Next,lithography is performed on the photoresist layer to define thenanochannels with a spacing that is larger than the optical resolutionof the readout system (See, e.g., FIG. 2 ). For example, a period of ˜1μm and a linewidth of 10- to 100-nm might be used. However, it will beappreciated that both smaller and larger periods and linewidths arereadily available. According to the embodiment shown in FIG. 2 ,interferometric lithography can be used to form the nanochannels, andthese dimensions are well within the capabilities of even one or twogeneration old lithographic tools, offering a ready extension to volumemanufacturing. Next, the antireflection layer is etched to expose thesubstrate. Colloidal nanoparticles (for example, silica nanoparticles)are then spin-coated on the photoresist pattern, thus depositing them ina layer-by-layer fashion first in the spaces between the photoresistlines to form the nanochannel sidewalls and finally extending over thephotoresist to form the nanochannel roofs.

As easily seen in FIGS. 3-5 , the nanoparticles form both the sidewallsand the roofs of the nanochannels, with the nanoparticles in the roofforming tortuous nanopores, which, should a sample be placed in thenanochannels, the DNA molecules would have to traverse the pores inorder to reach the roof and vice versa. According to some embodiments,50-nm-diameter silica nanoparticles are used, but both the size and thematerial structure are flexible. Capillary forces during depositionforce the nanoparticles (NP) into a hexagonal close-packed geometry. Asa rough estimate, this means that the spaces between nanoparticles are˜NP diameter/3 or ˜17 nm. The pores are complex, 3D paths, similar tothe spacings and open paths created when oranges are piled up in thelocal supermarket. However, it should be understood that the actualstructure is highly complex due to the significant dispersion innanoparticle sizes which is under the control of the nanochannelfabricator. For the purposes of the present disclosure, we refer to thespacings and open paths created by the nanoparticles as “tortuousnanopores.” In layer-by-layer deposition, steric effects due to the NPsize dispersion will create a range of nanopore sizes.

After spin-coating of the nanoparticles, the structure is then calcined(˜800° C. in an air ambient) to remove the remaining hydrocarbon films,to sinter the nanoparticles for additional mechanical strength, and toprepare the nanoparticles in a hydrophilic state that allows simplecapillary filling of the nanochannels with buffer/sample solution.

It will be readily understood that this is a very flexible nanochannelfabrication process. For silica nanoparticles, a simple dry etch stepallows for reservoirs with access to entry ports of the nanochannels andto provide electrodes for electrophoretic transport and stretching. Anadditional feature is the ability to stack several nanochannels witheither parallel or perpendicular nanochannel directions, simply byrepeating the above-process. See, e.g., FIG. 6 , which shows stackednanochannels. In addition to the nanochannel structure it is oftendesirable to introduce a secondary roof spaced away from the nanochannelroof. This ensures a flat surface for the buffer solution that movesfrom the nanochannels to the roof, provides a channel for flowing thetarget molecules away from the pore and allows an additional electrodefor controlling the translocation velocity.

Furthermore, a simple optical exposure before the spin coating stepenables the introduction of porous regions (barriers) along thenanochannels. As shown in FIG. 7-9 , these barriers can be used toaccumulate molecules of interest in the sample and localize thetranslocation of those molecules through the roof.

It will be appreciated that some applications that utilize theabove-described nanochannels would benefit from the ability tospecifically control the density of the nanopores in the nanochannelroof. For example, it might be desirable to reduce the density of thenanopores, so as to reduce or eliminate unwanted leakage or transport ofsamples through the roof and/or enabling the translocation,transportation, or identification of specific long chain molecules ofinterest including, for example, single stranded DNA (ssDNA), RNA andproteins. Accordingly, the present disclosure provides for the formationof tortuous nanopores that are formed in the nanochannel roof and whichcan be further decreased in size and density bystandard film depositionprocesses such as e-beam evaporation, sputtering, CVD and/or conformalatomic layer deposition (ALD). (The film deposition both closes some ofthe pores, reducing the density, and also decreases the sizes of theremaining pores allowing only a single long chain molecule to transit ata time.)

According to various embodiments, after the tortuous nanopores areself-assembled in the roof, the roof is partially sealed, by which it ismeant that some, but not all, of the externally accessible pores formedby the self-assembly and calcination of the nanoparticles are sealed.According to various embodiments, the pores may be sealed using eitherCVD, ALD, or a combination of both. For example, as described in greaterdetail below, a combination of CVD and ALD can be used to close thesmallest pores to prevent leakage or penetration of the sample throughthe roof, control pore density, and ensure compatibility with opticalresolution.

FIG. 10 is a high resolution SEM image of the porous roof; while FIG. 11is a high resolution SEM image of the porous roof after deposition of aplasma-enhanced CVD silicon nitride film. In FIG. 11 , the structure ispartially etched on the right side to form a reservoir and to provideaccess to the sides of the nanochannels. The white circles mark poresthat are just appearing as the etch progresses. The deposition tool usedfor the CVD puts down a porous layer, much like a blanket of snow, overthe NPs. This process can be tuned for varying degrees of film porosityby variation of the deposition conditions. An example of the processparameters used for the CVD deposition of silicon nitride include:T=300° C.; pressure of 600 mTorr; RF power of 50 W; and flow rates of[SiH₄] 30 ccm, [NH₃] 50 ccm, [N₂] 15 ccm. For the as-deposited roof,both the dispersion of the nanoparticle size and the dispersion ofnanopore sizes is evident. It is important to keep in mind that this isa tortuous nanopore, and the opening dimension is not necessarily thetightest constriction along the pore. As can be seen in FIG. 11 , theCVD film has largely covered the larger scale (˜10's of nm lineardimension) nanochannel pores, but some of the larger ones are beginningto be evident in the transition region between the as-deposited and theetched regions as marked by the white circles. The density anddimensions of these pores can be controlled by: 1) adjusting the NP sizedispersion, 2) the use of ALD before the CVD step to seal the majorityof the pores in the NP roof, 3) the use of different overlayers (eitherdielectric or metal prior to the active metal layer). Even in this firstexample, for which no optimization has been attempted, the nanoporedensity is close to the required separation of ˜λ to allow far-fieldresolution of the exit of each tortuous nanopore. The evaporation ofbuffer solution from the roof is evidence that the roof is porous, andthis evaporation time has been controlled over several orders ofmagnitude with the deposition and ALD steps outlined below. When a biasis applied across the nanochannels, particularly with a barrier, thereare isolated drops of fluid (and DNA) that emerge from the pores anddecorate the top of the roof (FIGS. 12-14 ). Note that the drops are notcontiguous, suggesting that the largest pores are well separated.

According to various embodiments, a nanopore structure with at least onemanufactured nanopore can be assembled on the roof of the tortuousnanopore structure. This could be a dense nanopore structure such as agraphene sheet or a sparse nanopore structure such as a nitride film inwhich nanopores are fabricated, e.g. by ion-milling, either before orafter application of the film to the tortuous nanopore structure. Sincethe goal is the read of long-DNA (as well as RNA and proteins)molecules, up to ˜50,000 bases or ˜10 μm of natural length, the tortuousnanopores structure reduces the DNA translocation speed through theconventional registering nanopore. (FIG. 15 ).

An alternative embodiment is to apply the tortuous nanopore to anexisting nanopore structure, for example an ion- or electron-milled porein a nitride film (FIG. 16 ). This could be done by applying ananoparticle suspension to one side of the pore and allowing it to dryto form the tortuous pathway for the DNA or similar long-chain molecule.The existing nanopore diameter can be adjusted so that ALD can be usedboth to restrict the translocation through the tortuous pathway throughthe nanoparticles and to decrease the diameter of the nanopore in thefilm. The pore in the film can be fabricated either before or after theformation of the tortuous pathway.

The evaporation rate from the pores provides a convenient measure of thepore density. For the as-fabricated nanochannels (prior to the CVD andALD treatments), when a drop of a buffer solution is introduced to thereservoirs, the fluid penetrates only a small distance, <1 mm, into thenanochannels before the fluid has evaporated (in low laboratoryhumidity). After the treatments discussed below, the penetrationdistance into the pores is increased to ˜1 cm.

In addition to adjusting the nanopore density and pore size, theaddition of a non-porous secondary optically transparent roof in closeproximity to the porous roof provides a means to adjust the localhumidity and hence control the evaporation rate out of the nanochannels.This roof can provide multiple enhancements to the device: 1) it canprovide a micro- or macro-flow channel for the buffer/molecular solutionon exiting the nanopores to allow removing them from the region of thepore and controlling the local humidity at the nanopore; 2) it canprovide an optical quality surface for far-field optical measurements;and 3) with the addition of a transparent electrode such as ITO, or agridded electrode structure, it can allow for further manipulation ofthe quasi-static electric fields in the vicinity of the tortuousnanopore to control the translocation velocity. (See e.g., FIG. 33 .)

Various approaches can be used to reduce the density of these pores andtherefore the evaporation rate from the channels. An exemplary approachutilizes a combination of SiO₂ CVD and atomic layer deposition (ALD)Evaporation can be estimated by the distance of liquid penetrationthrough the channels. If we put a drop of liquid on the porous roof wecan see that penetration of liquid through the channels is approximately1.5- to 2.5-mm and DNA solution easily penetrates through roofs with ˜15nm pores at the same distance. We observed the same penetration distanceof solution and DNA if we put the drop into the well. Chemical vapordeposition (CVD) of an 80- to 120-nm film of Si₃N₄ or SiO₂ over the roofreduces the evaporation and provides penetration of solution with DNA upto 3- to 4-mm. A further application of 10- to 20-nm thick atomic layerdeposition (ALD) of silica (SiO₂) or alumina (Al₂O₃) over the CVDdeposition reduces the roof pore size further and provides liquidpenetration up to 5-8 mm.

Other successful approaches utilize HfO₂ and Al₂O₃, which can be, forexample, deposited using standard semiconductor protocols for ALD. FIGS.15A-15C show the fluorescence from a drop of buffer/λDNA placed on topof the roof of an HfO₂ ALD nanochannel as it penetrates the roof. FIG.17A shows the drop approximately 15 seconds after it is placed on theroof. FIG. 17B shows the drop after 5 minutes and FIG. 17C shows thedrop after 10 minutes. The λDNA penetrates through the remaining poresin the roof and then spreads by diffusion along the nanochannels. Notethe long time scale of up to 10 min. for the evolution of thisdistribution. This suggests that: 1) the density of pores can besubstantially reduced with sufficient size for long dsDNA penetration,2) there is evidence that individual λDNA molecules are translocated,and 3) that the translocation time (without any applied bias) issufficiently long for sequencing as a result of the tortuous pathway.

Of course it will be appreciated that many, if not most, applications ofthe presently described device will implement a detection mechanism fordetecting the molecule of interest and that many suitable mechanisms arewell known and can be used with the presently-described device. However,it will also be understood that in particular, DNA sequencingapplications require very exacting detection methods that are capable ofachieving single base resolution, and thus the present disclosureprovides novel structures and enhance and enable detection at levelssuitable for DNA sequencing applications.

As is well known, a difficulty in achieving both single base sensitivityand resolution with far field optical techniques is associated with thelarge size of the photon, which can be focused to scales of ˜½ theoptical wavelength ˜300 nm, approximately two orders of magnitude largerthan the ˜0.3 nm linear dimension of the each of the DNA bases. This canbe addressed with a field enhancement structure that increases the localfield intensity in a small volume. Typically these field enhancementstructures are metals where excitation of surface plasma polaritonsleads to a strong field enhancement in a small local region. This is thebasis of surface enhanced Raman scattering, which has been well studiedfor many years.

As a first embodiment for localizing and enhancing the photon fields, ametal film can be deposited on the top of the nanochannel roof. If thefilm is deposited with a directional process such as, but not limitedto, electron beam evaporation, the film will be conformal with the finestructure of the roof, and in particular will have holes that arealigned with and on the scale of the tortuous nanopore exits. This is aself-aligned process, guided by the directional deposition and thetopology of the nanochannel roof, so no lithography step is required.

Alternative localized metal structures are: a dipole structure (twometal triangles pointed at each other with a small gap between them) ora “C” aperture (a metal loop with a small gap). Each of these produceslarge fields at the gap under optical excitation. These structures aredefined by a lithographic step, so they are appropriate for situationsin which the location of the nanopore is known a priori such as in thecase of manufactured nanopores produced by processes such aselectron-beam lithography or ion-beam milling.

As stated above, according to another embodiment, base-level opticalresolution can be provided by an engineered multi-levelmetal-insulator-metal (MIM) plasmonic structure that is self-assembledto the nanopores, providing a simple, inexpensive, and self-alignedfabrication process. The <1 nm insulator thickness provides thenecessary base-level resolution and the wide pore spacings allow forindependent far-field optical readout, providing a massively parallelsequencing capability. Furthermore, both labeled (fluorescence) andunlabeled (SECARS) optical readout mechanisms can be used with thissystem. This is related to the small gaps between aggregated colloidalAu and Ag nanoparticles which gives rise to single molecule Ramanscattering detection. Here, the gaps are engineered by to be aligned tothe exits of the tortuous nanopores on the roof of the nanochannels.

The MIM can be deposited by a combination of anisotropic and isotropicdeposition processes and can be self-aligned to the nanopores. Forexample, a thin metal film can first be deposited by e-beam evaporationor sputtering, a directional process that will not close the nanopore.Then a thin (e.g., ˜1 nm) insulator film can be deposited by atomiclayer deposition, a conformal deposition process that will furtherreduce the nanopore diameter. Finally, a second metal film can bedeposited by a directional process. This provides a self-aligned,massively parallel nanofabrication technology that bypasses the need forany high-resolution, ˜1-nm lithography and allows far-field opticalrecording of near-field processes with the necessary resolution. The MIMstructure both provides strongly enhanced electromagnetic fields,allowing single molecule detection, and the near-field nanoscaleresolution necessary to resolve individual bases in, for example,single-strand DNA (ssDNA). According to various embodiments, the motionof the sample through the nanochannels and nanopores is slowed by thetortuosity of the nanopores and can be further controlled by voltagesapplied to the channels, the MIM, and to control electrodes, which couldbe placed, for example, above the nanochannel roof.

The above-described technique can thus be used to form Raman “hot-spots”in those embodiments where a Raman spectroscopy-based detection methodis used. Surface Enhanced Raman Scattering (SERS) and surface-enhancedcoherent anti-Stokes Raman scattering (SECARS) are related techniquesthat offer the potential for both enhanced signal levels that havealready demonstrated single molecule level sensitivities. Bothtechniques rely on localized “hot-spots,” often at the intersticesbetween metallic particles (for example in colloidal aggregates). Thesehot-spots serve two essential purposes: 1) to ensure largeelectromagnetic fields (SERS, a two-photon process, scales as ˜E⁴ andSECARS, a four-wave mixing process, as ˜E⁸) providing the singlemolecule sensitivity and 2) to localize the interaction volume tosingle-base level dimensions—many orders-of-magnitude smaller thanλ³—providing the necessary single base resolution. This separation canbe engineered by the MIM structure described above. Thus, fieldenhancements of 30, which are quite reasonable for nanostructure metals,lead to Raman enhancements of ˜10⁶ and to SECARS enhancements of 10¹².Simply stated, Raman scattering is a mixing between an incident photonat frequency ω₁ and a molecular vibration at frequency ν, to provide ananti-Stokes signal at ω₁₊ν and a Stokes signal at ω₁−ν. The intensity,of the anti-Stokes signal is proportional to the occupation number ofthe molecular vibration, and is generally small at room temperaturewhere κT≤ν, where κ is Boltzman's constant and T the absolutetemperature (Kelvin). Coherently driving the excitation using twocoherent sources at frequencies ω₁ and ω₁−ν and detecting the signal atω₁+ν provides another enhancement of the Raman signal. This is known ascoherent anti-Stokes Raman scattering or CARS. By using a broadbandsecond (lower) laser frequency (for example a supercontinuum), we canprobe all four bases simultaneously. CARS is a four-wave mixing process(described by a third order nonlinear susceptibility, χ⁽³⁾). Analternate approach is to provide a source of phonons that directlyexcite the vibrational mode. These techniques maybe preferred in somecases as they are label-free and do not require any manipulation of theunknown DNA before sequencing. Raman spectra of each of the four DNAbases are well known, and offer readily separable signatures as shown inFIG. 18 (modified from a figure in reference 57). Fluorescence labelingtechniques have been demonstrated⁵⁸ and may be used as an alternatesequencing approach. Fluorescence, as spontaneous Raman scattering(SERS), involves two photons, and is enhanced (E⁴) and localized byplasmonic effects.⁵⁹

A schematic optical scheme is shown in FIG. 19 . According to anembodiment, the pump and Stokes excitation beams can be generated with aTi:sapphire laser and a nonlinear process such as an optical parametricoscillator or a supercontinuum generation scheme. The advantage of thesupercontinuum is that all four bases can be probed simultaneously(using, for example, dielectric filters to separate the anti-Stokeswavelengths). According to an exemplary arrangement, the laserilluminates through the nanochannel substrate, so that most of the pumplight is reflected by the MIM structure, simplifying the isolation ofthe SECARS signals. Since the device is probing a set of single bases,each localized to a resolution much smaller than the optical wavelength,there is no phase-matching requirement as in traditional CARS, and theradiation is emitted in a dipole radiation pattern. A judicious choiceof the illumination geometry suppresses the non-resonant four-wavemixing signal from the substrate and nanochannel materials, enhancingthe desired SECARS detectivity. According to an embodiment, the SECARSsignal can be collected with a high-NA objective and imaging onto eithera single detector for photon counting or onto a high-sensitivitycamera(s) for massively parallel multi-pore analysis. Since, by design,the pores are separated by more than the resolution limit of theobjective, the measurements for each channel are optically independent.

According to various embodiments, it is generally desirable for theRaman hot-spot to be sufficiently small and aligned with the exit of thetortuous nanopore so that the bases transit sequentially through thehot-spot. The presently described technique takes advantage of aself-aligned fabrication technique to ensure this overlap. Depositionprocesses such as e-beam evaporation and sputtering are directional, sothat when applied to the rough surface of our ALD coated nanoparticleroof; holes will form in a deposited metal film just at the porelocations, serving to define the locations of the hot spots. Additionallocalization can be enforced by fabricating a MIM structure. This can bedone using ALD to sequentially deposit a very thin dielectric layer(e.g., ˜0.5 to 1 nm) on the metal followed by a second metal layer,either with ALD or with directional deposition. The highly nonlinearSECARS process further reduces the extent of the hot-spot, providing therequired single base resolution. As a result of the stochasticdistribution of pore sizes, there might be some pores that allowtranslocation of more than one molecule, for example more than one ssDNAstrand simultaneously, or of residual dsDNA strands. Fortunately, thesecan be detected with temporal coincidences of reads of two bases in thesame location, and these pores can be ignored computationally, withoutrequiring any hardware modifications.

According to various embodiments, SECARS enables nanoscale-leveldiscrimination, even between bases in ssDNA. While the interactionleading to the Raman signature is confined in the near-field by thesmall dimensions of the apertures in the MIM and the spacing between thetwo metal films, the readout is in the far-field providing a massivelyparallel readout where each camera pixel can independently andsimultaneously address individual nanopores. In a fully engineeredsystem, long reads (>50 kilo-bases) with up to one million nanopores,separated by more than the resolution element of the observationmicroscopy, and a camera operating at 30 frames/s giving a throughput ofas much as 10¹¹ bases per hour is possible. Furthermore, the fluidicchip can be inexpensively produced and is designed to be fieldreplaceable.

As stated above, according to some embodiments, the presently disclosedapparatus can be used for the rapid and inexpensive separation,transportation, detection, and/or sequencing (referred to hereincollectively as “manipulation”) of nucleic acids, including, forexample, genomic DNA. According to this embodiment, each nanopore in theroof structure becomes an independent DNA translocation site that can beoptically resolved in parallel (˜1M per mm²). Moreover, it will beunderstood that a variety of potentials could be applied across thedevice to control the DNA translocation. For example, as described ingreater detail below, three or more potentials could be applied: alongthe nanochannels; between the nanochannels and the plasmonic readoutstructure; and above the plasmonic readout structure to provideexquisite control of the DNA translocation.

FIGS. 20A-20B depict of an exemplary method of DNA manipulationaccording to an embodiment of the disclosure. The DNA enters thenanochannel from the reservoir (not shown) on the left and is uncoiledby the dynamics in the nanochannel. Three sizes of silica nanoparticles(grey-scale differentiated) are shown to represent the dispersion in NPsize. The NPs form a close-packed quasi-hexagonal lattice disturbed bysteric effects as a result of the size dispersion, giving rise to anon-uniform set of tortuous pathways through the roof. An ALD process,represented by the dark borders on the NPs, closes the bulk of thenanopores (as evidenced by the dramatic reduction in evaporation ratethrough the roof following the ALD treatment), resulting in a density ofremaining nanopores that is compatible with far-field opticalresolution. The ALD can be controlled so that the majority of theremaining tortuous nanopores are sufficiently small that only a singlessDNA strand can pass through at a time. Finally, ametal-insulator-metal (MIM) structure (in expanded view in FIG. 20B)localizes the enhanced electromagnetic fields and provides single basemeasurement capability.

A typical experiment is illustrated in FIGS. 21-22 , where aDNA-containing solution is directly applied to the entrance of ananochannel array. A dyed (YoYo1) λDNA solution is applied to one sideof the channel and a buffer solution (without DNA) to the other end.FIG. 22 DNA penetration in the nanochannel. The image of FIG. 23 showsDNA entering the nanochannel when voltage is applied while FIG. 24 showsDNA moving out of the channel when the voltage is reversed.

In order to further investigate the influence of an electric field ondsDNA behavior in the herein described nanochannel, we have monitoredthe stretching of ds-DNA. The results demonstrate that an appliedelectric field causes the negatively charged dsDNA to migrate towardsthe positive contact. Some DNA molecules appear stuck in blockedchannels and accumulate. An applied electric field in the direction ofDNA movement stretched the molecules towards the positive electrode overmany 10's of μms (FIG. 25 ). Whereas, an electric field in the oppositedirection compressed the DNA to 2 μm (FIG. 26 ). The data shown in FIG.27 demonstrates the movement and stretching of DNA that can be achievedusing the herein described nanochannels by applying different potentialsacross the device. This enables the user to control the base spacing byselecting the appropriate direction and applied voltage of thepotential.

Those of skill in the art will be familiar with various methods forpreparing DNA libraries for sequencing. Exemplary methods andcommercially available kits that may be used alone or in combinationinclude the Qiagen DNA isolation kit for purifying dsDNA and the PromegaReadyAmp kit for ssDNA isolations. Long ssDNA isolation can be performedusing alkali treatment, neutralization of the pH, and maintenance of thesingle stranded stare with an optimized formamide buffer. Commercialkits are also available from Promega. Alternatively, asymmetric PCR canbe used to generate ssDNA. There are a number of publications thatdescribe the generation of ssDNA by asymmetric LATE-PCR, and this is arobust simple way to generate ssDNA. Alternatively, it may be desirableto first generate a sequencing library that is 10-20 kb and amplify thelibrary with an asymmetric primer ratio. The ssDNA can then be isolatedprior to application to the chip. The nanochannel chip can be run with50% formamide and at an elevated temperature to encourage ssDNA entryinto the channels. Due to entropic forces, the ssDNA should elongatealong the channel without secondary structure.

As an alternative to asymmetric PCR, it is also possible to use 50%biotinylated primers to capture amplified library fragments. To isolatessDNA, fragments can be captured with streptavidin coated beads. TheLibrary molecules that contain a single biotinylated primer and a singlenon-biotinylated primer can be eluted from the beads with a 0.1M NaOHwash. The pH of the supernatant is then neutralized, and the ssDNAfragments loaded into the nanochannels with a 50% formamide buffer. Yetanother approach involves exonuclease digestion in the nanochannels.Using this approach, it is possible to load very long (up to 50 kb)fragments with minimal library preparation.

As stated above, the presently described methods enable the productionof multileveled (i.e. tiered) nanochannels. FIGS. 28 and 29 depicttwo-tiered nanochannels engineered with the different tiers positionedin orthogonal directions. The use of two-tiered nanochannels allows forthe controllable introduction of an exonuclease, such as the T7exonuclease (5′->3′ exonuclease), in the vicinity of the nanopores inorder to digest a single strand of dsDNA. After the exonuclease hasdigested one of the strands, the ssDNA will be relatively resistant toexonuclease activity. This leaves the ssDNA in the nanochannel with the3′ end leading into the nanopore. The 3′ end can then advance through atortuous nanopore, enabling the sequence to be resolved using themethods described below.

According to various embodiments, the nanochannels described hereinenable dsDNA and/or ssDNA to move randomly through the tortuousnanopores in the roof of the nanochannels. In some embodiments it may bedesirable to optimize this translocation to ensure a desired spatialdensity (for example, ˜1 μm⁻¹) along the nanochannel to allow far-fieldoptical interrogation of individual nanopores.

Our preliminary data has shown that DNA moves out of the nanochannelsthrough the pores in the roof. The escape of DNA from the nanochannelspreferentially occurs when the channel is interrupted by a barrier asdemonstrated in FIGS. 30-32 , where FIG. 31 is an image showing DNAaccumulation at a barrier within a nanochannel and FIG. 32 is an imageshowing movement of DNA through a barrier. Accordingly, in someembodiments it may be desirable to construct barriers in the channels.In some embodiments it may be desirable for these barriers to havedifferent thicknesses. For example, one design can have thin barriers atthe beginning of the nanochannel and thicker barriers toward the end(see, e.g., FIG. 30 ).

According to various embodiments, multiple voltages can be applied toimpact the translocation. As shown in FIGS. 21-32 , voltages areroutinely applied along the nanochannels to control the position andconformation of the λDNA. In addition, and as shown in FIG. 33 , it maybe desirable to add a bias to the nanoplasmonic structure, and anindependent voltage to a transparent conducting layer [indium-tin-oxide(ITO)] located in the fluid volume above the plasmonic structure. All ofthese can be AC voltages with a DC bias. In electrical terms, this is afour-terminal device, giving us control over the translocation throughthe tortuous nanopores.

Turning to FIG. 34 , the present disclosure also contemplates a fullyintegrated lab-on-a-chip design in which a single device or “chip”fluidly connects the presently described structures via flowhomogenization channels which are able to connect the nanochannelsdescribed herein with structures, including, if needed, microchannelsized structures, intended to prepare the sample.

The specific methods and compositions described herein arerepresentative of preferred embodiments and are exemplary and notintended as limitations on the scope of the invention. Other objects,aspects, and embodiments will occur to those skilled in the art uponconsideration of this specification, and are encompassed within thespirit of the invention as defined by the scope of the claims. It willbe readily apparent to one skilled in the art that varying substitutionsand modifications may be made to the invention disclosed herein withoutdeparting from the scope and spirit of the invention. The inventionillustratively described herein suitably may be practiced in the absenceof any element or elements, or limitation or limitations, which is notspecifically disclosed herein as essential. The methods and processesillustratively described herein suitably may be practiced in differingorders of steps, and that they are not necessarily restricted to theorders of steps indicated herein or in the claims. As used herein and inthe appended claims, the singular forms “a,” “an,” and “the” includeplural reference unless the context clearly dictates otherwise.

What is claimed is:
 1. A method for manipulating target molecules usinga nanochannel covered with a porous roof comprising tortuous sealed andunsealed nanopores, the method comprising: introducing a buffer solutioninto the nanochannel; applying a first voltage potential across thenanochannel to move long chain target molecules in the buffer solutionthrough the nanochannel in a first direction; translocating the targetmolecules through the unsealed nanopores and onto the roof; anddetecting the target molecules that translocate through the unsealednanopores.
 2. The method of claim 1, further comprising: applying asecond voltage potential along the nanochannel by reversing the firstvoltage potential, such that the target molecules move through thenanochannel in a second direction opposite to the first direction; anddetecting the target molecules after applying the second voltagepotential.
 3. The method of claim 1, wherein applying a first voltagepotential comprises applying an alternating current (AC) voltagepotential having a direct current (DC) bias.
 4. The method of claim 1,wherein translocating the target molecules comprises applying a secondvoltage potential along the unsealed nanopores to control atranslocation velocity of the target molecules through the unsealednanopores.
 5. The method of claim 1, wherein: the roof further comprisesa field enhancement structure aligned with the unsealed nanopores; anddetecting the target molecules comprises detecting individual moietiesof the target molecules using surface-enhanced coherent anti-StokesRaman scattering (SECARS).
 6. The method of claim 1, wherein applying afirst voltage potential comprises moving the target molecules throughthe nanochannel to a barrier disposed in the nanochannel, prior totranslocating the target molecules through the unsealed nanopores. 7.The method of claim 1, wherein applying a first voltage potentialfurther comprises routinely applying multiple voltage potentials alongthe nanochannel.
 8. The method of claim 1, wherein applying a firstvoltage potential further comprises moving the target molecules acrossthe roof in the first direction.
 9. The method of claim 1, wherein: thetarget molecules comprise single-stranded or double stranded nucleicacids; and detecting the target molecules comprises detecting a basesequence of the target molecules.
 10. The method of claim 1, whereinintroducing a buffer solution comprises applying the buffer solution tothe roof, such that the buffer solution passes through the unsealednanopores and enters the nanochannel.
 11. The method of claim 1,wherein: the target molecules comprise single-stranded (SS) nucleicacids; introducing a buffer solution comprises introducing a buffersolution comprising double-stranded (DS) nucleic acid molecules into thenanochannel; and the method further comprises introducing an exonucleaseinto the nanochannel to digest the DS nucleic acid molecules and formthe target molecules.
 12. The method of claim 1, wherein: the targetmolecules comprise single-stranded (SS) nucleic acids; and introducing abuffer solution comprises introducing a buffer solution comprising theSS nucleic acids.
 13. The method of claim 1, wherein: the targetmolecules comprise single-stranded (SS) nucleic acids; and the buffersolution comprises a formamide buffer.
 14. A fluidic chip formanipulating target molecules, comprising: at least one nanochannel; aporous roof covering the nanochannel, the porous roof comprisingtortuous sealed and unsealed nanopores; and first and second electrodesdisposed on opposing ends of the nanochannel, the first and secondelectrodes configured to apply a first voltage potential along thenanochannel to move the target molecules through a buffer solutiondisposed in the nanochannel, wherein, each of the unsealed nanopores areconfigured such that only one of the target molecules can betranslocated through each unsealed nanopore at a time, and each of theunsealed nanopores have a density configured such that light emittedfrom the target molecules translocating through each unsealed nanoporecan be individually detected.
 15. The fluidic chip of claim 14, furthercomprising a transparent third electrode spaced apart from the porousroof by a space configured to receive a fluid volume.
 16. The fluidicchip of claim 15, wherein the third electrode comprises a transparentconductive material or a grid.
 17. The fluidic chip of claim 15, furthercomprising a field enhancement structure aligned with the unsealednanopores.
 18. The fluidic chip of claim 17, wherein field enhancementstructure and the third electrode are configured to apply a secondvoltage potential across the unsealed nanopores to control atranslocation velocity of the target molecules through the unsealednanopores.
 19. The fluidic chip of claim 18, wherein field enhancementstructure comprises a metal-insulator-metal structure.
 20. The fluidicchip of claim 14, further comprising a barrier structure disposed in thenanochannel and configured to direct at least some of the targetmolecules toward the unsealed nanopores.